The storage/release capability and chemical composition of synaptic vesicles will be compared in resting, moderately activated, and intensely activated electromotor nerve terminals of Torpedo in vitro. Stimulation induced alteration in vesicle morphology and its reversal during subseqent rest will be studied on the ultrastructural level by stereological analyses and by application of exogenous, high molecular weight tracers (dextrans, horseradish peroxidase). The releasable acetylcholine (ACh) pool in intact resting and stimulated terminals will be compared with the amounts of vesicular ACh isolated from these terminals; the recovery of the releasable pool and isolatable vesicular ACh after stimulation will be followed using radioactive precursors of ACh (acetate or choline). Concomitantly, miniature postsynaptic potentials (mPSPs) will be measured in order to learn how the frequency and amplitude of mPSPs varies as a function of the alteration of transmitter stores in terminals. Vesicles isolated from resting and stimulated tissue will be purified and their ACh storage capacity will be compared. Protein composition of vesicles isolated from resting and stimulated terminals will be analyzed by sodium dodecylsulfate polyacrylamide slab gel electrophoresis. Combined ultrastructural, electrophysiological, and biochemical results will be used to define how the cycling of vesicles affects their storage/release capability and hence the functional state of presynaptic terminals. The relevance of results obtained on electroplaque synapses to central and peripheral mammalian cholinergic synapses will be assessed in appropriate parallel experiments on rat brain synaptosomal preparations and/or rat neuromuscular junctions.